I can detect my Flag-HA tagged protein with anti-HA but not anti-Flag - (May/02/2014 )

I have a human protein that is tagged at the N-terminus with a 1x flag tag followed by an HA tag. After transient transfection of this vector into 293Ts and protein harvest, I can detect the protein by western blot with an anti-HA antibody but not the anti-flag antibody (Sigma Flag M2). Has anyone had issues with a 1x flag tag? I know the anti-flag antibody works because I can detect another protein that is flag tagged. 

Kozak - 1xFlag - HA - ORF

Any idea what is going on here?

Thanks!

I stopped using M2 long ago because of such problems. I couldn't use it in IP experiments because it was too sensitive to detergents in my lysis buffer.

I've just started using M2.  What antibody do you use now?

Missle on Mon May 5 15:53:28 2014 said:

I've just started using M2.  What antibody do you use now?

I am sure your question is in reference to curtis but I thought I would give some feedback as well. HA tag and anti-HA works well for me. This was the first time trying Flag and M2. 

I dont use Flag anymore. His works better for me. I think Sigma-Aldrich introduced a new Flag system already called 3x. Im not sure if they are still selling the original Flag. I think only Sigma has the right to sell this. Because I never saw other companies sell it.